Recovery of feed products from stillage by refermentation



Patented May 6, 1952 UNITED STATES PATENT OFFICE RECOVERY OF FEED PRODUCTS FROM STILLAGE BY REFERMENTATION Original application March 30, 1945, Serial No. 585,698. Divided and this application October 3, 1947, Serial No. 777,708

8 Claims. 1

This invention relates to the recovery of feed by-products from stillage from a primary alcohol fermentation, as for example grain fermentation stillage or slop, and more particularly to a new process of treatment of such stillage to increase the vitamin content thereof, thereby increasing the nutritive value of of the recoverable feed. The present application is a division of our copending application U. S. Serial No. 585,698, filed March 30, 1945, now abandoned.

Recovery of feed by-products from distillers residues is commonly practiced at the present time. In many instances only the screenable solid portion of the residue is recovered, the liquid portion being run off into streams as waste. In other instances both the insoluble and soluble solids are recovered together, while in still other instances various fractions may be recovered separately, yielding distillers dried grains, distillers dried grains with solubles, and distillers dried solubles. These materials are high quality, high protein feeds, and, in the case of solubles, are also rich in water-soluble B-complex vitamins.

We have found that the stillage from a primary alcohol fermentation can, under suitable conditions, be refermented with the yeast or yeastlike organism Ashlrya gossypii to produce desired vitamins from the residual carbohydrates and other constituents left in the stillage. Concentration and drying of the refermented vitaminenriched stillage by customary methods gives a final product of high vitamin content which, can be advantageously used as an ingredient of mixed feeds for livestock and poultry or as a vitamin carrying material for preparing human foods.

We have further found that if a source of nitrogen is added to the stillage the micro-organism will also synthesize protein and thus increase this valuable constituent in the final product. Likewise our work has shown that the synthesis of desirable vitamins can be increased by adding a small quantity of easily fermentable carbohydrate to the stillage.

Briefly, the process as developed by us consists in adjusting the pH and temperature of the previously sterilized stillage to values favorable to microbial fermentation, inoculation of the medium with the prepared micro-organism, fermentation with controlled aeration, and then concentrating first to a thick syrup, and then to a freeflowing solid material by evaporating and drying procedures customarily used for recovering distillers by-products.

From the following description of the invention it will be noted that, in the main, the novel features reside in control of the flora through sterile handling of the sterile stillage, sterilization at an acid pH which accomplishes considerable hydrolysis, careful acidity and nutrilite control at the beginning and during refermentation, regulating the length of time allowed for refermen. tation to give the maximum vitamin yield, and selection of Ashb'ya gossym'i as the micro-organism capable of refermentating the stillage in a relatively short period of time to increase the vitamin content and yield a nutritionally more balanced feed. I

The carbohydrate that is left in the stillage from a primary alcohol fermentation, as, for example, grain alcohol fermentation, is non-fermentable by ordinary distillers yeast to alcohol. However, our experimentations have shown that Ashbya gossypii will ferment the carbohydrate and other compounds remaining in such stillage to very materially increase the B-complex vitamin content, including not only the commonly known but also the less well known members of that group, as well as other vitamin-like complexes and accessory materials of known physiological importance but unknown chemical structure.

The preferred process will be described with reference from time to time to the accompanying flow sheet. Hot stillage from the still I, after passing through the screen 2 is pumped to a centrifuge 3 where more suspended solids not removed by the screen are taken out. The clarifled stillage passes from the centrifuge 3 to a storage tank 4. If preferred, the centrifuge may be by-passed, the screened stillage being taken directly to storage tank 4. From the latter the stillage is pumped to a sterilizer 5. Complete sterilization can be efiected by introducing steam at ten pounds pressure for about one hour, or by pumping the stillage through a heated conduit. It would also be possible to sterilize the 6.0 to 7.5 is satisfactory. The acid present in the stillage may be neutralized with an hydroxide of an alkali or alkaline earth metal, as for example sodium hydroxide, potassium hydroxide, calcium hydroxide and the like. Likewise alkaline salts such as sodium carbonate, potassium carbonate and calcium carbonate may be used as neutral izing agents. Certain ammonium compounds will also serve as neutralizing agents in this connection as for example ammonium hydroxide and ammonium carbonate. The stillage is neutralized in the sterilizer 5, the alkalizing agent being supplied from a chemical storage tank 6, and is then pumped to a cooler l' where it is cooled to a temperature optimum for refermentation. .A temperature of between approximately 70 and 90 degrees Fahrenheit is preferred, this being the temperature at which the stillage should be maintained at the time of inoculum addition and during refermentation. stillage is pumped to the fermenter 8.

If preferred, sterilization and initial pH adjustment may be performed in the fermenter 8 in which case the stillage would be pumped directly from the screen 2, or from the centrifuge 3 if that is used, to the fermenter 8 which will have been previously sterilized by steam treatment. We might also explain that if desired the pH may be adjusted before sterilization. However we prefer to sterilize at the lower pH and then adjust the pH to the higher value because additional nutrients are released by sterilization at the lower pH.

It might be appropriately mentioned at this point that if, during the course of refermentation, excess alkalinity or acidity develops after the initial pH adjustment it is necessary to add an acidifying or alkalizing agent, as the case may be, to bring the pH back into the favorable range for vitamin synthesis. The alkalizing agent may be any one of the aforementioned compounds described as suitable for initially neutralizing the acid present in the stillage. As an acidifying agent acids such as hydrochloric, phosphoric and acetic, or acid salts such as acid phosphates and acid sulphates may be used. Such agents may be supplied to the stillage in the fermenter from a chemical storage tank 9.

The sterilized stillage, appropriately adjusted as to pI-I and temperature, having reached the fermenter 8, a suitable amount of a culture of the micro-organism Ashbya gossypz'i is then introduced from a pure culture machine I0, and aeration is started by introducing air which has previously been sterilized, as for example by passage through tubes of sterile cotton, over white hot iron, treatment with ultraviolet light, or the like. In the flow sheet the source of air supply is shown at H, the air sterilizer at l2 and the air measuring device at 13. The volume of air should be the optimum for the micro-organism.

The volume of inoculum and the age of the inoculum depend on the micro-organism used. In the case of Ashbya gossypiz, a 48 hour culture and one percent by volume of inoculum have been found satisfactory. The pure cultures used are grown on standard laboratory media, and aeration may be supplied by introducing a stream of sterile air through a tube, or by mechanical shaking.

The time required for completion of the refermentation may vary considerably. It may run as high as 60 hours, or more, or as low as 10 to 12 hours. When refermentation is completed the stillage is evaporated by standard methods to a thick syrup and then dried to a solid. For example it may be pumped from the fermenter 8 to an evaporator is where it is evaporated under a vacuum of about 20 millimeters of mercury to a From the cooler I the such as acetamide and urea.

4 thick syrup. It is then passed to a drum dryer l5 where it is dried to a free flowing solid.

Although it can be evaporated at the pH of refermentation, we find that we get better results if we acidify the refermented stillage to a pH of approximately 5.0 before evaporating. By so doing we find that we get less foam and a lighter colored final product. A mineral acid such as hydrochloric or phosphoric may be used for this acidification and may be supplied from a suitable compartment in the chemical storage tank 9.

Because of the manner in which the vitamins are synthesized right in the stillage prior to evaporation and drying, the final dried product possesses characteristics which render it most useful as a feed ingredient. Because the vitamins are evenlyfdispersed in the concentration process the final dried product is free from any tendency of settlingl'out or separation of the vitamins such as occurs when crystalline vitamins are mixed with a flaky or ground material 1. e. simply suspended between particles. In the case of riboflavin this dispersion within the particles protects the'riboflavin from light, thus greatly decreasing the destructive efiect of light. This internal dispersion of the vitamins also decreases oxidative deterioration due to contact with air. For these reasons the product is very stable and can be stored for long periods with but small loss of vitamin potency. The product is also superior to one obtained by simply mixing with known crystalline vitamins because the microorganism used greatly increases the content, not only of the known but also of the less well-known, vitamins and vitamin-like complexes of recognized physiological importance but whose chemical structure, being unknown, are not yet capable of being synthesized in the laboratory, and hence are not available for fortification by mixing.

Reference has already been made to protein synthesis as a step in our novel process. We prefer to supplement the vitamin synthesis with protein synthesis and we have found that this may be accomplished in the same process of refermentation by adding to the stillage prior to inoculation and refermentation a small amount of a nitrogen-containing nutrient material such as an ammonium compound, for example ammonium hydroxide and ammonium carbonate, or an amino compound, such as glycine, or an imino compound, such as occurs in proline, or an amide Such compounds, we find, are synthesized to proteins, polypeptides or amino acids, by the same micro-organisms which synthesize components of the vitamin B complex. and thus increase the protein value of the final feed in proportion to the amount of nitrogen added. Where the nitrogen containing material is also an alkalizing agent, as for example ammonium hydroxide, it also serves as a neutralizing agent for the excess acid in the stillage, thus making unnecessary the addition of another alkalizing agent to control the pH. The nitrogen-containing material is preferably added to the stillage while it is in the sterilizer 5, and before sterilization. It may be supplied from a suitable compartment in the chemical storage tank 6, or from a compartment in the tank 9 if the stillage is run directly to the fermenter from the screen 2 or the centrifuge 3.

Also, as has already been mentioned, We prefer to increase the vitamin synthesis by the addition to the stillage of a small quantity of an easily fermentable carbohydrate, as for example addition of 2% or less by weight of cane molasses or sugar. Carbonhydrate'addition is not necessary to effect vitamin improvement but such addition will increase the improvement. Such supplemental nutrients are preferably added before sterilization, and may be supplied from a suitable compartment in the chemicalstorage tank 6 or from a compartment in the tank 9 if the stillage is run directly to the fermenter from the screen 2 or the centrifuge 3.

If preferred, the whole stillage can be refermented before screening to yield a soluble fraction of greatly increased vitamin potency. In that case the refermented stillage would then be screened and also centrifuged, if desired, before being concentrated to a solid in the manner previously described. l

The following example, illustrating a typical procedure we have carried out, and the resultant product obtained therefrom, will serve to give a clearer understanding of the invention:

Two hundred milliliters of centrifuged thin stillage (3.8% solids) obtained from a primary fermentation mash consisting of 79.20% corn, 11.00% rye and 9.80% barley malt, were placed in a 500 'ml. Erlenmeyer flask and the pH adjusted to 7.2 with 10% sodium hydroxide using a Beckman pH meter. .The flask and appropriate connections were sterilized in an autoclave at pounds pressure for 30 minutes. After cooling, the media was inoculated with 2 milliliters of a 48 hour broth culture of Ashbg a gossypi'i. The

contents of the flask were aerated by a stream of sterile air (one bubble per second through a 3 millimeter capillary tube). The refermentation was carried out for 84 hours at a temperature of 72 de rees F.

The material (3.7% solids) was condensed in vacuo and dried under and infra-red ray lamp. The comparison of the dried product with the original material when dry is as follows:

Dry Dry Original ag g Mammal Material McgJgram McgJgram Riboflavin 15 75 Pantothcnic acid 69 Niacin 105 234 steam per pound of water is required. Thus more of the drying process can be carried out in the evaporators when drying our refermented. stillage than is possible when concentrating primary stillage. This is no doubt due to additional protein denaturing in the refermentation process which makes the material less sticky, that is, less viscous than primary stillage.

It will be understood that the product resulting from our refermentation process is primarily intended for use as an accessory product in poultry and animal feeds as an auxiliary source of the B complex vitamins. Because of its vitamin potency it will be preferably used in relatively small percentages as a supplementary ingredi- 6 cut. 'While the additional protein which can be synthesized during the refermentation will also add valuable supplementary protein to the feed, such protein'source is a secondary consideration in such feeds because only a relatively small percentage of the new product will be needed in a ration to give the desired additional vitamin concentration in the mixed feed. For example in a poultry ration the main constituents comprise ground corn, certain wheat by-products, soybean meal, a small quantity of some type of meat scrap, etc. Assaying of these constituents indicates a deficiency in the B complex vitamins as well as the fat soluble vitamins, namely A and D. The aforementioned constituents contain certain quantities of the nutritionally essential I vitamins but not in sufiicient quantities to givea nutritionally balanced ration. Therefore fish or other oils containing vitamins A and D are added; likewise supplemental sources of the B complex vitamins are added, and it is in this last connection that our new product is particularly useful. The addition of 2% to 5% of these vitamin carrying materials to the aforementioned cereals or cereal by-products gives a nutritionally bal-v anced ration, that is a ration which is balanced as to protein, fat, etc., as well as its content of the various vitamins.

What we regard as new and desire to secure by Letters Patent is 1. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the step which comprises subjecting the stillage to the fermentative action of the ascomycete Ashbya gossypiz' under controlled aeration.

2. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the steps of which comprise subjecting the stillage to the fermentative action of the ascomycete Ashbya gossypii under controlled aeration and while maintaining the pH and temperature of the fermentation mixture at values favourable to growth of said ascomycete.

3. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the steps which comprise adjusting the pH of the stillage to between approximately 6.0 and 7.5 and the temperature to between approximately 70 and Fahrenheit and then subjecting it to the fermentative action of the ascomycete Ashbya. gossypii under controlled aeration and while maintaining the temperature within the aforesaid limits.

4. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the steps which comprise adding an alkalizing agent selec'ted from the group consisting of the hydroxides and alkaline salts of the alkali and alkaline earth metals to bring the pH to between approximately 6.0 and 7.5, adjustin the temperature to between approximately 70 and 90 Fahrenheit and then subjecting it to the fermentative,

action of the ascomycete Ashbya gossypii under controlled aeration and while maintainin the temperature within the aforesaid limits.

5. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins 01 the B complex and other growth-stimulating substances, the steps of which comprise adding to the stillage a small amount of a nitrogen-containing material and then subjecting it to the fermentative action of the ascomycete Ashbya gossypz'i under controlled aeration.

6. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the steps which comprise adding to the stillage a nitrogen-containing alkalizing agent to bring the pH to between approximately 6.0 and 7.5, adjusting the temperature to between approximately 70 and 90 Fahrenheit and then subjecting it to the fermentative action of the ascomycete Ashbya gossypii under controlled aeration and while maintaining the temperature within the aforesaid limits.

7. In the treatment of stillage from a primary alcohol fermentation to increase the content of water soluble vitamins of the B complex and other growth-stimulating substances, the steps which comprise addin to the stillage a small ercentage of an easily fermentable carbohyrate, and then subjecting it to the fermentative action of the ascomycete Ashbya gossypz'i under controlled aeration.

8. The method of treating grain alcohol stillage to increase the content of B complex vitamins and other growth stimulating substances which comprises the steps of sterilizing the stillage, inoculatingit with the yeast Ashbya gossypii and fermenting the inoculated stillage while supplying air to the fermenting medium.

CLAIR S. BORUFF. LEONARD STONE. JACOB C. BAUERNFEIND. JOHN C. GAREY.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 1,532,858 Bacon Apr. 7, 1925 2,098,200 Stiles Nov. 2, 1937 2,202,161 Miner May 28, 1940 2,374,503 Rudert Apr. 24, 1945 2,400,710 Piersma May 21, 1946 2,445,128 Tanner July 13, 1948 2,447,814 Novak Aug. 24, 1948 OTHER REFERENCES Raffy: Comptes Rendus, T126, December 4, 1937, pp. 875 to 877.

Schopfer: Helvetica Chemica, Acta V, XXVII, pp. 1017 to 1032; p. 1019 relied upon.

Guilliermond: Comptes Rendus, T200, May 6, 1935, pp, 1556 to 1558.

Wicker-ham: Arch. of Biochem, vol. 9, No. 1, January 1946. P1195 to 98. 

1. IN THE TREATMENT OF STILLAGE FROM A PRIMARY ALCOLHOL FERMENTATION TO INCREASE THE CONTENT OF WATER SOLUBLE VITAMINS OF THE B COMPLEX AND OTHER GROWTH-STIMULATING SUBSTANCES, THE STEP WHICH COMPRISES SUBJECTING THE STILLAGE TO THE FERMENTATIVE ACTION OF THE ASCOMYCETE ASHBYA GOSSYPII UNDER CONTROLLED AERATION. 